The contractile responses of isolated and activated bovine retinal microarteries (BRA) (diameter, 204 +/- 4 microns; n = 48) to acetylcholine (ACh) were studied. These responses depended on the nature of the activating agent. The ACh relaxed BRA activated by prostaglandin F2 alpha (PGF2 alpha) and circumferential stretching in a dose-dependent manner but had no significant effect on K(+)-activated BRA. The effects of ACh also depended on the degree of BRA activation: the stronger the PGF2 alpha-induced contractions, the weaker the relaxation produced by ACh. In equivalent PGF2 alpha-induced contractions, the ACh effects were reproducible. The muscarinic antagonist, atropine, reversed the relaxation caused by ACh of PGF2 alpha-activated BRA. Physostigmine, an inhibitor of acetylcholinesterase, did not potentiate or prolong the relaxant action of ACh. Selective removal of the BRA endothelium (by gassing the BRA lumen, checked by scanning electron microscopy) blocked the relaxation caused by ACh of PGF2 alpha-induced contractions and unmasked a constricting action of ACh. This suggests that, in BRA with functional endothelium, the direct constricting effects of ACh on smooth muscle are masked by the more potent dilating activity, mediated by endothelial muscarinic receptors. Acetylcholinesterase was not found in BRA.